U.S. patent number 6,548,296 [Application Number 09/113,692] was granted by the patent office on 2003-04-15 for methods for identifying human cell lines useful for endogenous gene activation, isolated human lines identified thereby, and uses thereof.
This patent grant is currently assigned to Roche Diagnostics GmbH. Invention is credited to Johannes Auer, Michael Brandt, Reinhard Franze, Konrad Honold, Hans Koll, Ulrich Pessara, Anne Stern.
United States Patent |
6,548,296 |
Stern , et al. |
April 15, 2003 |
Methods for identifying human cell lines useful for endogenous gene
activation, isolated human lines identified thereby, and uses
thereof
Abstract
The invention concerns human cells which, due to an activation
of the endogenous human EPO gene, are able to produce EPO in an
adequate quantity and purity to enable a cost-effective production
of human EPO as a pharmaceutical preparation. Furthermore the
invention concerns a process for the production of such human
EPO-producing cells, DNA constructs for the activation of the
endogenous EPO gene in human cells as well as a process for the
large-scale production of EPO in human cells.
Inventors: |
Stern; Anne (Penzberg,
DE), Brandt; Michael (Iffeldorf, DE),
Honold; Konrad (Penzberg, DE), Auer; Johannes
(Penzberg, DE), Koll; Hans (Weilheim, DE),
Franze; Reinhard (Penzberg, DE), Pessara; Ulrich
(Weilheim, DE) |
Assignee: |
Roche Diagnostics GmbH
(Mannheim-Waldhof, DE)
|
Family
ID: |
27217986 |
Appl.
No.: |
09/113,692 |
Filed: |
July 10, 1998 |
Foreign Application Priority Data
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Jul 23, 1997 [EP] |
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97112640 |
Dec 1, 1997 [EP] |
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97121073 |
Dec 3, 1997 [DE] |
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197 53 681 |
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Current U.S.
Class: |
435/366; 435/325;
435/367; 435/69.1; 435/70.1 |
Current CPC
Class: |
C07K
14/505 (20130101); C12N 15/10 (20130101); C12N
15/625 (20130101); C12P 21/005 (20130101); A61K
38/00 (20130101); C07K 2319/02 (20130101); C07K
2319/036 (20130101); C07K 2319/75 (20130101) |
Current International
Class: |
C07K
14/435 (20060101); C07K 14/505 (20060101); C12P
21/00 (20060101); C12N 15/10 (20060101); C12N
15/62 (20060101); A61K 38/00 (20060101); C12P
021/06 (); C12P 021/04 (); C12N 005/00 (); C12N
005/02 () |
Field of
Search: |
;435/6,69.1,91.4,455,463,325,366,367,375,320.1,70.1,70.3
;530/350,351,412,413,416,417 ;930/90 |
References Cited
[Referenced By]
U.S. Patent Documents
Foreign Patent Documents
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411678 |
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Dec 1985 |
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EP |
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232034 |
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Jan 1987 |
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EP |
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267678 |
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Sep 1987 |
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EP |
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747485 |
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Nov 1990 |
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EP |
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9009627 |
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Dec 1990 |
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WO |
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9209627 |
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Nov 1992 |
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WO |
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9311704 |
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Dec 1993 |
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WO |
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9603377 |
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Mar 1995 |
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WO |
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95060454 |
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May 1995 |
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WO |
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9500696 |
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Dec 1995 |
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WO |
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9601988 |
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May 1996 |
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WO |
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WO96/35718 |
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Nov 1996 |
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WO |
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WO 96/35718 |
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Nov 1996 |
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WO |
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Other References
Yanagi et al. 1989. Recombinant human erythropoietin produced by
Namalwa cells. DNA 8:419-427.* .
Mucke, S. et al., "Suitabiloity of Epstein-Barr virus-based
episomal vectors . . . ", Gene Therapy, vol. 4, pp. 82-92, Feb.
1997.* .
Stellwagen. Gel Filtration. in Guide to Protein Purification,
Methods in Enzymology. 1990. vol. 182, pp. 317-328.* .
Krystal, et al. Purification of Human Erythropoietin to Homogeneity
By a rapid Five Step Process, Blood 87(1):71-79 (1986). .
Simonsen, et al, "Isolatioin and expression of an altered mouse
dihydrofolate reductase-cDNA," Proc. Natil. Acad Sci
USA:80:2495-2499 (1983). .
Recny, et al, "Structural characterization of natural Human Urinary
And Recmbinant DNA-derived Erythroprotein" JH. Biol. Chem
2645(5):17156-17163 (1987)..
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Primary Examiner: Yucel; Remy
Assistant Examiner: Katcheves; Konstantina
Attorney, Agent or Firm: Fulbright & Jaworski, LLP
Claims
We claim:
1. An isolated, human recombinant cell produced by endogenous gene
activation which comprises a CMV promoter sequence in operable
linkage with the endogenous gene which encodes a protein, wherein
10.sup.6 of said cells produce at least 200 ng of said protein per
24 hours, wherein said protein is erythropoietin and wherein said
endogenous gene is not amplified.
2. The isolated, human recombinant cell of claim 1, wherein
10.sup.6 of said cells produce from about 200 to about 3000 ng of
said protein per 24 hours.
3. An isolated, human recombinant cell produced by endogenous gene
activation wherein said human recombinant cell comprises a
heterologous promoter in operable linkage with said endogenous
gene, wherein said endogenous gene encodes erythropoietin, wherein
said CMV promoter and said endogenous gene are amplified and
wherein 10.sup.6 of said cells produce at least 1000 ng of
erythropoietin per 24 hours.
4. The isolated, human recombinant cell of claim 3, wherein
10.sup.6 of said cells produces from 1000-25,000 ng of said protein
per 24 hours.
5. The isolated, human recombinant cell of claim 1 or claim 3,
wherein said cell is culturable in serum-free medium.
6. The isolated, human recombinant cell of claim 1 or claim 3,
wherein said cell is an HT 1080 cell, a HeLa S3 cell, or a Namalwa
cell.
7. A process for purifying erythropoietin comprising: (i) culturing
the isolated human recombinant cell of claim 1 in a culture medium,
under conditions favoring production of erythropoietin encoded by
said endogenous gene, to produce a cell supernatant containing
erythropoietin, (ii) passing said cell supernatant over an affinity
chromatography medium to isolate a fraction containing
erythropoietin, (iii) passing the fraction obtained in (ii) over a
hydroxyapatite column to isolate a fraction containing
erythropoietin, and (iv) concentrating the fraction obtained in
(iii) over a reverse phase, high performance liquid chromatography
medium to isolate erythropoietin therefrom.
8. The process of claim 7, further comprising passing the fraction
obtained in (i) over a hydrophobic interaction chromatography
medium to obtain an erythropoietin containing fraction prior to
contacting said fraction to the hydroxyapatite of (iii).
9. The process of claim 7, wherein said affinity chromatography
medium is blue Sepharose.
10. The process of claim 8, wherein said hydrophobic interaction
chromatography medium is butyl Sepharose.
11. The process of claim 7, wherein said concentrating comprises
exclusion chromatography.
12. The process of claim 11, comprising excluding molecules less
than 10 kD from those larger than 10 kD.
13. An isolated, human recombinant cell which comprises a CMV
promoter sequence in operable linkage with an endogenous gene which
encodes a protein, wherein 10.sup.6 of said cell produces at least
200 ng of said protein per 24 hours, wherein said endogenous gene
is not amplified, wherein the isolated human recombinant cell is
produced by endogenous gene activation of an endogenous gene in a
human cell that comprises more than two chromosomes containing the
endogenous gene and wherein the human cell does not express the
endogenous gene prior to endogenous gene activation, wherein said
human cell undergoes 5 doublings over a period of 14 days or less
in suspension culture, 5 doublings over a maximum period of
fourteen days in a serum free culture medium and is capable of
correctly glycosylating the protein.
14. An isolated, human recombinant cell which comprises a CMV
promoter sequence in operable linkage with an endogenous gene which
encodes a protein, wherein 10.sup.6 of said cell produces at least
200 ng of said protein per 24 hours, wherein said endogenous gene
is not amplified, wherein the isolated human recombinant cell is
produced by endogenous gene activation of an endogenous gene
encoding erythropoietin (EPO) in a human cell that comprises more
than two chromosomes containing the endogenous gene encoding EPO
and wherein the human cell does not express the endogenous gene
prior to endogenous gene activation.
15. An isolated, human recombinant immortalized cell which
comprises a CMV promoter sequence in operable linkage with an
endogenous gene which encodes a protein, wherein 10.sup.6 of said
cell produces at least 200 ng of said protein per 24 hours, wherein
said endogenous gene is not amplified, wherein the isolated human
recombinant immortalized cell is produced by endogenous gene
activation of an endogenous gene in a human immortalized cell that
comprises more than two chromosomes containing the endogenous gene
and wherein the human cell does not express the endogenous gene
prior to endogenous gene activation.
Description
FIELD OF THE INVENTION
The invention relates to methods for identifying human cells useful
in endogenous gene activation, in order to produce human proteins.
The invention also involves processes for manufacture of proteins,
such as human proteins in cells identified in this manner, as well
as the isolated cells so identified.
BACKGROUND AND PRIOR ART
The production of human proteins by endogenous gene activation in a
human cell line is known. See, e.g., WO 93/09222, WO 94/12650 and
WO 95/31560 for example, describing the production of human
erythropoietin and other human proteins in human cell lines by
endogenous gene activation.
These references do not advise, however, as to what criteria have
to be observed when selecting cells used to produce human proteins.
In fact, the methods described in these references do not ensure
high yield and contaminant free production of human protein. In
fact, only low yields of human proteins are achieved following the
above cited references.
As noted, supra, human erythropoietin is described in these
references as a protein, the production of which is desired. A
discussion of erythropoietin and the art relating to its production
is set forth here, although it is to be borne in mind that
erythropoietin production is simply exemplary of the invention,
which is in no way limited to this protein.
Erythropoietin ("EPO" hereafter) is a glycoprotein which stimulates
the production of red blood cells. EPO is present only in very low
concentrations in the blood plasma of healthy persons, so it is not
possible to prepare large amounts via purification of plasma.
EP-B-0148 605 and EP-B-0205 564, incorporated by reference,
describe the production of recombinant human EPO in Chinese Hamster
ovary, or "CHO" cells. The EPO described in EP-B-0148 605 has a
higher molecular weight than EPO purified from urine and is not
O-glycosylated. The EPO from CHO cells described in EP-B-0 205 564
is available in large amounts and in pure form, but it is derived
from non-human cells. Further, the production yield of CHO cells is
also often relatively limited.
As alluded to supra, it is known that human EPO ("hEPO") can be
isolated from the urine of patients with aplastic anemia (Miyake et
al., J. Biol. Chem. 252 (1977), 5558-5564). A seven-step process is
disclosed in this reference, which involves, inter alia, ion
exchange chromatography, ethanol precipitation, gel filtration and
adsorption chromatography. In this process an EPO preparation with
a specific activity of ca. 70,000 U/mg protein is obtained in a 21%
yield. The disadvantages of this process and other processes for
isolating urinary EPO include obtaining the starting material in
adequate amounts and with a reproducible quality. Furthermore, the
purification of hEPO from urine is difficult and even a purified
product is not free of urinary impurities.
GB-A-2085 887, incorporated by reference, describes a process for
the production of human lymphoblastoid cells which are able to
produce EPO in small amounts. It is not possible to economically
produce EPO of the desired quality using the human lymphoblastoid
cells disclosed herein.
WO 91/06667 as noted supra, describes a process for the recombinant
production of EPO. In this process the endogenous EPO gene is
operatively linked to a viral promoter in a first process step by
homologous recombination, in primary human embryonic kidney cells.
The recombined DNA is then isolated from these cells. In a second
step, the isolated DNA is transformed into CHO cells, and the
expression of EPO in these cells is analyzed. There is no
indication that it is possible to produce EPO in human cells.
WO 93/09222 describes the production of EPO in human cells. In this
process relatively high levels of EPO production, i.e., up to
960,620 mU/10.sup.6 cells/24 hours is achieved using human
fibroblasts which have been transfected with a vector containing
the complete EPO gene. These transfected cells contain an exogenous
EPO gene which is not located at the correct EPO gene locus,
leading to problems with respect to the stability of the cell line.
The reference does not discuss constitutive EPO production.
Moreover, there is also no information about whether the EPO
produced is of sufficient quality for, e.g., pharmaceutical
use.
Activation of the endogenous EPO gene in human HT1080 cells is also
described in this reference, but production of only 2,500
mU/10.sup.6 cells/24 hours (corresponding to ca. 16 ng/10.sup.6
cells/24 hours) is found. Such low production levels are unsuitable
for economic production of a pharmaceutical preparation.
WO 94/12650 and WO 95/31560, incorporated by reference, describe
that a human cell with an endogenous EPO gene activated by a viral
promoter is capable, after amplification of the endogenous EPO
gene, of producing EPO in an amount of up to ca. 100,000
mU/10.sup.6 cells/24 hours (corresponding to ca. 0.6 .mu.g/10.sup.6
cells/24 hours). Even this amount is still not sufficient for the
economic production of a pharmaceutical preparation.
As indicated, supra, the cells and cell lines disclosed in the
literature relating to endogenous gene activation, while useful,
are by no means totally satisfactory. It has now been found,
however, that it is possible to identify and to isolate cells and
cell lines which will be useful in high yield production of
proteins, following endogenous gene activation via, e.g.,
homologous recombination. Hence, one aspect of the invention is a
method for identifying such cells and cell lines. A second feature
of the invention are the cells and cell lines so identified. Yet a
third feature of the invention is the use of the cells and cell
lines for the production of proteins, using these cells and cell
lines. How these and other aspects of the invention are achieved
will be seen from the disclosure which follows.
BRIEF DESCRIPTION OF THE FIGURES
FIG. 1 shows a schematic representation of the amplification of
homology regions of the EPO gene from the region of the 5'
untranslated sequences, exon 1 and intron 1 and the primers of PCR
product 1 (upper strand=SEQ ID NO: 1; lower strand=SEQ ID NO: 2)
and PCR product 2 (upper strand=SEQ ID NO: 3; lower strand=SEQ ID
NO: 4);
FIG. 2 shows a schematic representation of a plasmid which contains
EPO homology regions from the region of the 5' untranslated
sequences, exon 1 and intron 1;
FIG. 3 shows a schematic representation of a gene activation
sequence which contains the Rous-sarcoma virus promoter (RSV), the
neomycin phosphotransferase gene (NEO), the early polyadenylation
region of SV40 (SVI pA), the early SV40 promoter (SVI), the
dihydrofolate reductase gene (DHFR), an additional early SV40
polyadenylation region and the cytomegalovirus immediate-early
promoter and enhancer (MCMV);
FIG. 4a shows the construction of the EPO gene targeting vector
p176;
FIG. 4b shows the construction of the EPO gene targeting vectors
p179 and p187;
FIG. 4c shows the construction of the EPO gene targeting vector
p189 (DSM 11661);
FIG. 4d shows the construction of the EPO gene targeting vector
p190;
FIG. 4e shows the construction of the EPO gene targeting vector
p192;
FIG. 5 shows a schematic representation of the construction of EPO
cDNA with signal sequence mutations;
FIG. 6a shows the hybridization of cellular DNA with a probe from
the CMV region of the gene cassette shown in FIG. 3; lanes 1 to 4
each show DNA from human cells, cleaved with the restriction
enzymes AgeI and AscI; lane 1: EPO-producing HeLa S3 cell amplified
with 1,000 nM methotrexate (MTX); lane 2: EPO-producing HeLa S3
cell amplified with 500 nM MTX; lane 3: EPO-producing HeLa S3 cell
without amplification; lane 4: HeLa S3 cell without activated EPO
gene; lane 5: digoxigenin-labelled length marker. The size of the
hybridizing fragment in lanes 1 to 3 is ca. 5,200 bp; and
FIG. 6b shows the hybridization of a probe from the coding region
of EPO with DNA of human cells. Lane 1 shows digoxigenin-labelled
length markers; lanes 2 to 4 show DNA from human cells, cleaved
with the restriction enzymes BamHI, HindIII and SalI; lane 2: EPO
producing HeLa S3 cell amplified with 500 nM MTX (length of the
band produced by the non-activated endogenous gene: 3,200 bp;
length of the copy of the EPO gene activated by gene targeting:
2,600 bp); lane 3: DNA from a non-amplified EPO-producing HeLa S3
cell; lane 4: DNA from a HeLa S3 control cell.
DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS
As indicated, supra, one feature of this invention is a method for
identifying and isolating cells and cell lines, preferably human
cells and cell lines, which are useful in the production of
endogenous proteins via gene activation, preferably by homologous
recombination. In the method, a cell or cell line, preferably
human, is first tested to determine if the desired gene of
interest, which will generally correspond to the naturally
occurring nucleotide sequence, is present. The details of how this
is determine and are set forth, infra. If it is determined that the
cell or cell line contains the desired sequence, then it is
determined if a population of the cell or cell line doubles, at
least five times, in a period of 14 days, when incubated in
suspension culture. If the cell or cell line satisfies this
criterion, then it is tested to see if a population of the cell or
cell line will double, at least five times over 14 days if grown in
serum free culture medium. If the cell or cell line satisfies this
criterion, then it will be useful in activation of the gene of
interence, via, e.g., homologous recombination.
It is preferred that the starting materials are immortalized human
cell lines. These are preferred because they have known advantages
with respect to culturability which need not be summarized here.
Such cell lines, it must be borne in mind, can exhibit mutations in
their genomes, so the presence of the desired sequence must be
confirmed. The polymerase chain reaction, or "PCR," for example,
can be used to determine this, following standard protocols.
Assuming the cell or cell line possesses the desired sequence, it
is tested for its ability to be cultured. Suspended cells are
easier to ferment and the fermentation can be more easily adapted
to larger dimensions, such as large fermenters with a volume of 10
to 50,000 liters. Consequently, the selected cells should either be
cells which are known to be culturable in suspension or they should
be readily adaptable to suspension culture. One determines this by
culturing the cells for 14 days while stirring continuously. If the
population of the cells doubles at least five times within this
period, they are regarded as being suitable for the next step of
the invention. The number of population doublings can be determined
by periodic determinations of the cell count, e.g., by mechanical
cell counting or by measuring the optical density of the cell
suspension.
A further important feature of the cells or cell lines of the
invention is the culturability in serum-free medium. Since the
purification of proteins from serum-free cell cultures is
considerably simpler, and in serum-free culture there is no risk of
contamination with animal pathogens such as viruses, it should be
possible to culture the selected cells in serum-free culture. To
determine this, the cells are cultured for 14 days at a density of
1 to 10.times.10.sup.5 cells per ml in culture vessels containing
serum-free medium (e.g., RPMI 1640 containing insulin, transferrin
and selenide). If the population of the cells doubles at least five
times during this culture period, determined as above, they are
regarded as being suitable for serum-free culture.
A further important feature of the invention, representing a
preferred embodiment, is the generation time. The selected cells
should exhibit a high proliferation in media such as DMEM, 10%
fetal calf serum or RPMI 1640 containing 10% fetal calf serum,
i.e., within one week in culture their population should double 10
to 256 times, preferably 64 to 128 times. To determine this, the
cells are seeded in culture plates at a concentration of 0.1 to
10.times.10.sup.5 cells per ml, preferably 0.5 to 2.times.10.sup.5
cells per ml and the cell count is determined every two to three
days with the aid of a cell counting chamber after or without
trypsinization. Cells which have a sufficiently short generation
time are particularly suitable for large-scale production of human
proteins by endogenous gene activation.
A further preferred embodiment of the invention is the absence of
detectable endogenous expression, i.e., transcription and
translation, of the target gene. Preferably, those cell lines are
selected for endogenous gene activation which have essentially no
or no endogenous expression of the target gene. To determine this,
the cells are seeded at a cell density of 0.01 to 2.times.10.sup.6
cells/ml, preferably 0.5 to 1.times.10.sup.6 cells/ml of culture
medium. After a predetermined time period, e.g., 24 hours, the cell
supernatant is removed, the cells are discarded and the content of
the target protein is determined in the cell supernatant by means
of known test procedures, e.g., ELISA. In the case of EPO, the
detection limit may be 10 pg/EPO/ml. Cells which yield less than 10
pg protein when seeded at 10.sup.5 cell/ml are regarded as
non-producers and are particularly suitable.
It is especially preferred that the target gene exhibit polysomy in
the selected cell. The presence of more than two chromosomal copies
of the target gene in the cell significantly increases the yield of
protein, following homologous recombination. The cells Namalwa
(Nadkarni et al, Cancer 23 (1969), 64-79) or HeLa S3 (Puck et al.,
J. Exp. Med. 103 (1956), 273-284) which possess three copies of
chromosome 7 have proven to be particularly suitable for the
production of EPO, whose gene lies on chromosome 7. Further
examples of cell lines that contain more than one copy of
chromosome 7 are the colon adenocarcinoma cell line SW-480 (ATCC
CCL-288; Leibovitz et al., Cancer Res. 36 (1976), 4562-4567), the
malignant melanoma cell line SK-MEL-3 (ATCC HTB 69; Fogh and Tremp,
in: Human Tumor Cells in vitro, pp 115-159, J. Fogh (ed.), Plenum
Press, New York 1975), the colon adenocarcinoma cell Colo-320 (ATCC
CCL-220; Quinn et al., Cancer Res. 39 (1979), 4914-4924)), the
melanoma cell line MEL-HO (DSM ACC 62; Holzmann et al., Int. J.
Cancer 41 (1988), 542-547) and the kidney carcinoma cell line A-498
(DSM ACC 55; Giard et al., J. Natl. Cancer Inst. 51 (1973),
1417-1423).
The number of chromosomes in the genome of a cell line can be
determined by using DNA probes which are specific for the
respective chromosome or/and the locus of the target gene.
It is especially preferred that the cell line used for endogenous
gene activation correctly glycosylates the desired target protein.
A human cell line which synthesizes the target protein with a
glycosylation pattern which is comparable to and is preferably
indistinguishable from the naturally occurring target protein,
especially in the number of sialic acid residues is particularly
preferred. The ability to correctly glycosylate can be tested by
transiently transfecting the cell of interest with, e.g., a vector
such as an extrachromosomal vector which contains the desired
target gene under the control of a promoter that is active in the
cell. After transient expression of the target gene the cell
supernatant or/and the cell lysate is analyzed by isoelectric
focusing. The presence of correct glycosylation can be easily
determined. For example, non-glycosylated EPO, i.e., recombinant
EPO from E. coli cells, has activity comparable to glycosylated EPO
in in vitro experiments. However, in in vivo experiments,
non-glycosylated EPO is considerably less effective. In order to
determine whether a starting cell line is able to produce EPO with
correct glycosylation, comparison can be made to urinary EPO, or
with recombinant EPO from CHO cells, which is known to have a form
of glycosylation which is active in humans and whose glycosylation
is substantially identical to that of urinary EPO. Glycosylation is
preferably compared by isoelectric focusing.
It is preferred that the cell line be free of infectious
contamination, i.e., infectious viral particles or mycoplasmas. The
examination for the presence of viral contamination can be carried
out by means of cell culture, in vivo analyses or/and detection of
specific viral proteins.
Cells or cell lines so identified can be used in a process for the
production of human proteins by endogenous gene activation of a
human cell line, which meets the above listed criteria.
The process according to the invention can be used to produce
substances such as EPO, thrombopoietin (TPO), colony-stimulating
factors such as G-CSF or GM-CSF, proteins which influence blood
coagulation such as t-PA, interferons such as IFN-.alpha.,
IFN-.beta. or IFN-.gamma., interleukins such as IL-1 to IL-18,
chemokines such as MIP, neurotrophic factors such as NGF or BDNF,
proteins which influence bone growth such as IFG-BPs, hedgehog
proteins, tumor growth factors such as TFG-.beta., growth hormones
such as hGH, ACTH, enkephalins, endorphins, receptors such as
interleukin or insulin receptors in soluble or/and membrane-bound
forms and other protein binding proteins. The process is
particularly preferably used to produce EPO.
Endogenous gene activation can be carried out according to known
methods. Preferably, it comprises the steps: (a) providing human
starting cell lines which contain at least one copy of an
endogenous target gene with the desired nucleic acid sequence and
which have been identified as being suitable for the expression of
the target gene via the processes described supra, (b) transfecting
the cells with a DNA construct comprising: (i) two flanking DNA
sequences which are homologous to regions of the target gene locus
in order to allow homologous recombination, (ii) a positive
selection marker gene, (iii) optionally a negative selection marker
gene, (iiii) optionally an amplification gene and (v) a
heterologous expression control sequence which is active in the
human cell, (c) culturing the transfected cells under conditions in
which a selection takes place for the presence of the positive
selection marker gene and optionally for the absence of the
negative selection marker gene, (d) analyzing the cells that can be
selected according to step (c), (e) identifying the cells producing
the desired target protein and (f) optionally amplifying of the
target gene in the selected cells.
The DNA construct used to produce the cell producing the desired
human protein contains two flanking DNA sequences homologous to
regions of the target gene locus. Suitable flanking sequences can
be selected for in accordance with the methods described in WO
90/11354 and WO 91/09955. The flanking sequences preferably are at
least 150 bp long. The homologous DNA sequences are particularly
preferably selected from the 5' region of the target gene, e.g.,
5'-untranslated sequences, signal-sequence-coding exons and introns
located in this region, e.g., exon 1 and intron 1.
The positive selection marker gene can be any selection marker gene
suitable for eukaryotic cells which leads to a selectable phenotype
when expressed. Antibiotic resistance, auxotrophy etc. are examples
of this. A particularly preferred positive selection marker gene is
the neomycin phosphotransferase gene.
A negative selection marker gene can be present, but is not
required. Examples of these include HSV thymidine kinase gene,
expression of which leads to death in the presence of a selection
agent. The negative selection marker gene is located outside of the
two flanking homologous sequence regions, and is preferably
downstream of the 3' homology region.
If amplification of the endogenously activated target gene in the
human cell is desired, the DNA construct is provided with an
amplification gene. Examples of suitable amplification genes are
the dihydrofolate reductase gene, the adenosine deaminase gene, the
ornithine decarboxylase gene, etc. A particularly preferred
amplification gene is the dihydrofolate reductase gene, more
particularly a gene coding for a dihydrofolate reductase arginine
mutant which has higher sensitivity for the selective agent
(methotrexate) than the wild-type gene. See Simonsen et al., Proc.
Natl. Acad. Sci. USA 80 (1983), 2495, incorporated by
reference.
It is especially preferred that the DNA construct used for the
endogenous gene activation contains a heterologous expression
control sequence that is active in a human cell. The expression
control sequence comprises at least a promoter and preferably
further sequences which improve expression, such as enhancer
sequences. The promoter can be a regulatable or constitutive
promoter. The promoter is preferably a strong viral promoter, e.g.,
an SV40 or a CMV promoter. The CMV promoter/enhancer is
particularly preferred.
Endogenous genes as used herein refers to an endogenous gene not
modified in the region which encodes the desired, mature
polypeptide.
In accordance with the invention, one can isolate a human cell
which contains a copy of an endogenous EPO gene in operative
linkage with a heterologous expression control sequence that is
active in the human cell and which is capable of producing at least
200,ng EPO/10.sup.6 cells/24 hours without prior gene
amplification. Such human cells preferably produce 200 to 3000 ng
EPO/10.sup.6 cells/24 hours and are most preferably able to produce
1000 to 3000 ng EPO/10.sup.6 cells/24 hours, under appropriate
culture conditions.
Human cells which contain several copies of an endogenous EPO gene,
each in operative linkage with a heterologous expression control
sequence that is active in the human cell and are able to produce
at least 1000 ng EPO/10.sup.6 cells/24 hours, are also a facet of
the invention. Such human cell lines preferably produce 1,000 to
25,000 ng EPO/10.sup.6 cells/24 hours and most produce 5,000 to
25,000 ng EPO/10.sup.6 cells/24 hours under appropriate culture
conditions.
The human cells and cell lines of the invention can be of any cell
type, provided that they are culturable in vitro, in serum free
medium, particularly in suspension culture. In this manner, it is
possible to produce EPO in large fermenters of about 1,000 liters
or more.
Preferably, immortalized cells such as HT 1080 cells (Rasheed et
al., Cancer 33 (1974), 1027-1033), HeLa S3 cells (Puck et al., J.
Exp. Med. 103 (1956), 273-284), Namalwa cells (Nadkarni et al.,
Cancer 23 (1969), 64-79) or a cell derived therefrom are used.
HT1080 cells, HeLa S3 cells, and cells derived therefrom are
especially preferred.
The cells of the invention are characterized by linkage of the
endogenous EPO gene to a heterologous expression control sequence
that is active in the human cell. The expression control sequence
comprises a promoter, and preferably, further sequences that
improve expression, such as enhancer sequences. The promoter can be
a promoter that can be regulated, or a constitutive promoter. The
promoter is preferably a strong viral promoter, e.g., an SV40
promoter, or a CMV promoter. The CMV promoter/enhancer is
particularly preferred.
Furthermore, in order to optimize expression of proteins such as
EPO, it is preferable that the endogenous gene in the human cell
that is in operative linkage with the heterologous promoter have a
signal peptide coding sequence which is different from the natural
signal-peptide-coding sequence, and preferably codes for a signal
peptide with a modified amino acid sequence. A
signal-peptide-coding sequence which codes for a signal peptide
sequence that is modified in the region of the four first amino
acids. These first four amino acids satisfy the formula: Met Xaa
Xaa Xaa (SEQ ID NO: 5)
wherein the first Xaa is Gly or Ser, the second Xaa is Ala, Val,
Leu, Ile, Ser or Pro, and the third Xaa is Pro, Arg, Cys, or His,
with the proviso that this four amino acid sequence is not Met Gly
Val His (SEQ ID NO: 6).
Particularly preferred are: (a) Met-Gly-Ala-His (SEQ ID NO: 7), (b)
Met-Ser-Ala-His (SEQ ID NO: 8), (c) Met-Gly-Val-Pro (SEQ ID NO: 9)
or (d) Met-Ser-Val-His (SEQ ID NO: 10)
The sequence of the first four amino acids of the signal peptide is
especially preferably Met-Ser-Ala-His.
A further aspect of the present invention is a DNA construct useful
for activating an endogenous EPO gene in a human cell comprising:
(i) two flanking DNA sequences which are homologous to regions of
the human EPO gene locus, selected from 5' untranslated sequences,
exon 1 and intron 1, in order to allow homologous recombination
wherein a modified sequence is present in the region of exon which
codes for a four amino acid sequence as described supra: (ii) a
positive selection marker gene, (iii) a heterologous expression
control sequence which is active in a human cell and (iv)
optionally an amplification gene.
A further aspect of the present invention is a DNA construct for
activating an endogenous EPO gene in a human cell comprising: (i)
two flanking DNA sequences which are homologous to regions of the
human EPO gene locus and are selected from 5' untranslated
sequences, exon 1 and intron 1 in order to allow a homologous
recombination, (ii) a positive selection marker gene, (iii) a
heterologous expression control sequence which is active in a human
cell wherein the distance between the heterologous expression
control sequence and the translation start of the EPO gene is not
more than 1100 bp and (iv) optionally an amplification gene.
Surprisingly it has been found that when the EPO signal sequence is
modified and/or when the distance between the heterologous
expression control sequence and the translation start of the EPO
gene is shortened, optimized expression is obtained. The distance
between the promoter of the heterologous expression control
sequence and the translation start of the EPO gene is preferably
not more than 1100 bp, particularly preferably not more than 150 bp
and most preferably not more than 100 bp. A particularly preferred
example of a DNA construct that can be used according to the
invention is the plasmid p189 (DSM 11661) or a plasmid derived
therefrom.
Yet a further aspect of the present invention is a process for the
production of human EPO in which a human cell according to the
invention is cultured in a suitable medium under conditions in
which production of EPO takes place and the EPO is isolated from
the culture medium. A serum-free medium is preferably used as the
medium. The cells are preferably cultured in suspension. The
production preferably takes place in a fermenter in particular in a
large fermenter with a volume of for example 10-50,000 liters.
The isolation of human EPO from the culture medium of human cell
lines preferably comprises the following steps: (a) passing the
cell culture supernatant over an affinity chromatography medium and
isolating the fractions containing EPO, (b) optionally passing the
fractions containing EPO over a hydrophobic interaction
chromatography medium and isolating the fractions containing EPO,
(c) passing the fractions containing EPO over hydroxy-apatite and
isolating the fractions containing the EPO and (d) concentrating
and/or passing over a reverse phase (RP)-HPLC medium.
Step (a) of the purification process comprises passing the cell
culture supernatant, which can optionally be pretreated, over an
affinity chromatography medium.
Preferred affinity chromatography media are those on which a blue
dye is coupled. A particularly preferred example is blue-Sepharose.
After elution from the affinity chromatography medium the eluate
containing EPO is optionally passed over a hydrophobic interaction
chromatography medium. This step is expedient if a culture medium
with a serum content >2% (v/v) is used. If a culture medium is
used with a low serum content, e.g., 1% (v/v) or a serum-free
medium, this step can be omitted. A preferred hydrophobic
interaction chromatography medium is butyl-Sepharose.
The eluate from step (a) or--if used--step (b) is passed over
hydroxyapatite in step (c) of the process according to the
invention and the eluate containing EPO is subjected to a
concentration step or/and a reverse phase HPLC purification step.
The concentration is preferably carried out by exclusion
chromatography, such as membrane filtration and the use of a
medium, such as a membrane with an exclusion size of 10 kD has
proven to be favorable.
An isolated human EPO with a specific activity of at least 100,000
U/mg protein in vivo (normocytaemic mouse) is obtainable by the
process according to the invention which is free of urinary
impurities and may or may not differ in its glycosylation from
recombinant EPO from CHO cells. Preferably, the EPO of the
invention has specific activity of at least 175,000, more
preferably at least 200,000, and up to about 400-450,000 IU/mg of
protein. The human EPO which is obtainable by the process according
to the invention can contain .alpha.-2,3-linked or/and
.alpha.-2,6-linked sialic acid residues. When EPO obtained from
cells which contain an endogenously activated EPO gene was
examined, the presence of .alpha.-2,3-linked and .alpha.-2,6-linked
sialic acid residues was found. Furthermore, it was found that
human EPO according to the invention has a content of less than
0.2% N-glycol-neuraminic acid relative to the content of N-acetyl
neuraminic acid.
The purity of the human EPO obtained in accordance with the
invention is at least 90%, more preferably at least 95% and most
preferably at least 98% relative to the total protein content. The
total protein content can be determined by reverse phase HPLC,
e.g., with a Poros R/2H column.
Human species of proteins, such as EPO can be obtained by the
process according to the invention which differ in their amino acid
sequence. Thus for example using mass spectrometric analysis
(MALDI-MS) it was found that a human EPO can be isolated from HeLa
S3 cells wherein the isolated EPO consists essentially of a
polypeptide with a length of 165 amino acids which is formed by
C-terminal processing of an arginine residue. Up to about 15% of
the recovered EPO may be 166 amino acids in length, depending on
the culture conditions used. In addition a human EPO can also be
obtained which consists of a polypeptide with a length of 166 amino
acids, i.e., a non-processed EPO. Human EPO from Namalwa cells was
isolated which contained a mixture of polypeptides with a length of
165 and 166 amino acids.
Such proteins, such as human EPO, can be used as an active
substance for a pharmaceutical preparation which can optionally
contain further active substances as well as standard
pharmaceutical auxiliary, carrier and additive substances.
A further aspect of the present invention is an isolated nucleic
acid molecule which codes for a human EPO with a modified sequence
in the region of the first 4 amino acids of the signal peptide as
described supra.
Genomic DNA and cDNA are both a part of the invention.
How these and other features of the invention are achieved will be
seen in the examples which follow.
EXAMPLE 1
1. Culture
Cell lines were seeded at a concentration of 0.1-5.times.10.sup.5
per ml, preferably 0.5-2.times.105 cells per ml in culture plates
containing DMEM and 10% FCS or RPMI 1640 and 10% FCS and the cell
count was determined every two to three days during culture with a
counting chamber, with or without trypsinization, in a medium
recommended for the respective cells and under suitable conditions.
Cells which exhibited 16 to 256 population doublings, preferably 64
to 128 population doublings, within one week culture were assessed
as positive (+, ++ or +++).
1.2 Ability to Culture in Suspension
In order to determine the ability to culture the cells in
suspension, samples were cultured for 14 days at 37.degree. C. and
7% CO.sub.2 while stirring continuously in medium as above with and
without the addition of serum, e.g., fetal calf serum. Cells which
exhibited at least 5 population doublings during this phase were
assessed as being suitable (+) for a suspension culture.
1.3 Ability to Culture in Serum-free Medium
In order to determine whether the cells could be cultured in serum
free medium, they were cultured under conditions according to 1.1
for 14 days at a density of 1-10.times.10.sup.5 cells/ml in culture
vessels in the basic medium (without serum supplementation). Cells
whose population doubled at least 5 times during this period
(determined by cell counting) were assessed as being suitable (+)
for serum-free culture.
1.4 Determination of Endogenous Expression of the Target Gene
In order to determine whether the target protein is produced in the
selected cells, the cells were seeded at a cell density of 0.01 to
2.times.10.sup.6 cells/ml preferably 0.5 to 1.times.10.sup.6
cells/ml culture medium for 24 hours. The cell culture supernatant
was removed later, cells were discarded, and the content of cell
protein in the cell culture supernatant was determined by known
methods, e.g., by a specific immunoassay for the respective
protein.
In the case of EPO the content was determined by means of an ELISA.
For this, streptavidin-coated microtitre plates were coated with
biotinylated anti-EPO antibodies and incubated with a solution
containing protein (1% w/v) to block unspecific binding. Then, 0.1
ml samples of culture supernatant was added and incubated
overnight. After washing, peroxidase-conjugated monoclonal anti-EPO
antibodies were added for 2 hours. The peroxidase reaction was
carried out in a Perkin Elmer photometer at 405 nm using ABTS as a
substrate.
The detection limit for EPO in this test was 10 pg EPO/ml. Cells
which produced less than 10 pg EPO/ml when seeded at 10.sup.6
cells/ml were assessed as non producers and as suitable (+).
1.5 Determination of the Number of Copies of the Target Gene
In order to examine the number of copies of the target gene in the
cell line, human genomic DNA was isolated from ca. 10.sup.8 cells
and quantified following Sambrook et al., Molecular Cloning, A
Laboratory Manual (1989), Cold Spring Harbor Laboratory Press.
After cleaving the DNA with restriction enzymes, e.g., Agel and
AscI or BamHI, HindIII and SalI the DNA fragments were separated
according to size by agarose gel electrophoresis and finally
transferred onto a nylon membrane and immobilized.
The immobilized DNA was hybridized with a digoxigenin-labelled DNA
probe which was specific for the locus of the target gene or for
the chromosome on which the target gene was located and washed
under stringent conditions. The specific hybridization signals were
detected with the aid of standard chemiluminescence methodologies
using radiation-sensitive films.
1.6 Determination of the Nucleic Acid Sequence of the Target
Gene
The genomic DNA was isolated from ca. 10.sup.7 cells using a
commercially available DNA isolation kit.
A pair of PCR primers was used to amplify the target gene. The
sequences of these primers were complementary to sequences which
flank the coding region of the target gene. This enabled the
amplification of the entire coding region of the target gene.
The PCR product was either directly subjected to sequence analysis
or cloned into a vector and subsequently sequenced. Sequencing
primers which are complementary to sequences from the intron
regions of the target gene were used, so that the sequences of the
exon regions of the target gene could be obtained completely. The
sequencing was carried out on an automated sequencer using
commercially available materials and instructions.
1.7 Determination of the Glycosylation Pattern
In order to determine the glycosylation pattern of EPO, the cell
lines to be tested were transfected with the plasmid pEPO 227 which
contains a 4 kb HindIII/EcoRI fragment of the human EPO gene
sequence under the control of the SV40 promoter (Jacobs et al.
Nature 313 (1985), 806; Lee-Huang et al. Gene 128 (1993), 272). The
cells were transfected in the presence of lipofectamine using a
commercially available reagent kit according to the manufacturer's
instructions. The EPO content was determined by ELISA in the cell
supernatant isolated 2 to 5 days later.
The cell supernatant was concentrated and compared to known EPO
products by isoelectric focusing (Righetti P. G., in: Work T. S.,
Burdon R. H. (ed.), Isoelectric focusing: Theory, methodology and
applications, Elsevier Biomedical Press, Amsterdam (1983)). Human
cells which yielded a comparable glycosylation pattern to known EPO
products, e.g., urinary EPO were assessed as suitable (+).
1.8 Determination of Viral Contamination
1.8.1. Analyses by Means of Cell Culture
In order to determine viral contamination of human cell lines
tested lysates of the cells were incubated with a detector cell
line in order to detect cytopathic effects. Hemadsorption analyses
were also carried out.
In order to produce the lysate, a suspension of 10.sup.6 cells was
lysed in 1 ml buffer by a rapid freeze-thaw process. The cellular
residue was separated by centrifugation and the supernatant was
added to the detector cell lines. HepG2 (ATCC HB-8065; Nature 282
(1979), 615-616), MRC-5 (ATCC-1587) and Vero (ATCC CCL-171; Jacobs,
Nature 227 (1970), 168-170) cells were used as detector cell lines.
Polio, SV, and influenza type viruses were used as a positive
control. Detector cell lines that had been cultured without lysate
were used as negative control. In order to determine cytopathic
effects the detector cell lines were regularly examined over a
period of at least 14 days.
For hemadsorption analysis, Vero cells which had been incubated
with the cell lysates or with the controls were admixed, after 7
days, with erythrocytes from chickens, pigs or humans. An
attachment of the erythrocytes to the monolayer of cultured cells
indicates viral contamination of the cultures.
1.8.2. In vivo Analysis
Lysates of the cell lines to be examined were prepared as stated in
1.8.1 and injected intraperitoneally or intracerebrally into
newborn mice (0.1 ml per injection). The mice were observed with
regard to morbidity and mortality over a period of 14 days.
1.8.3 Specific Detection of Viral Proteins
The presence of specific viral proteins, e.g., Epstein-Barr virus
proteins (nuclear protein or capsid antigen) was tested by adding
human serum of EBV-positive bands to immobilized cells of the cell
line to be tested. The virus antigens were then detected by adding
complement and the corresponding anti-human complement C3
fluorescein conjugate (to detect the nuclear antigen) or via
anti-human globulin fluorescein (to detect the capsid antigen).
The human cell lines HepG2, HT 1080, Namalwa, HeLa, and HeLaS3 were
tested as described. The results are set forth at Table 1.
It can be seen from table 1 that cell lines HT1080, Namalwa and
HeLa S3 satisfied the required and preferred criteria with Namalwa
and HeLa S3 being particularly preferred.
EXAMPLE 2
Cloning of EPO Homology Regions
Homology regions of the EPO gene were amplified by PCR using human
placenta genomic DNA. For this, two PCR products were prepared from
a 6.3 kB long homology region from the region of the
5'-untranslated sequences of the EPO gene, exon 1 and intron 1 (cf
FIG. 1). The primers used to prepare the PCR product 1 had the
following sequences: 5'-CGCGGCGGAT CCCAGGGAGC TGGGTTGACC GG-3' (SEQ
ID NO: 1) and 5'-GGCCGCGAAT TCTCCGCGCC TGGCCGGGGT CCCTCAGC-3' (SEQ
ID NO: 2). The primers used to prepare the PCR product 2 had the
following sequences: 5'-CGCGGCGGAT CCTCTCCTCC CTCCCAAGCT GCAATC-3'
(SEQ ID NO: 3) and 5'-GGCCGCGAAT TCTAGAACAG ATAGCCAGGC TGAGAG-3'
(SEQ ID NO: 4).
The desired segments were cut out of the PCR products 1 and 2 by
restriction cleavage (PCR product 1: HindIII, PCR product 2:
HindIII and EcoRV) and cloned into the vector pCRII which had been
cleaved with HindIII and EcoRV. The recombinant vector obtained in
this manner was named 5epopcr1000 (cf. FIG. 2).
EXAMPLE 3
Construction of EPO Gene Targeting Vectors 3.1 A gene activation
sequence which contains the NEO gene, the DHFR gene and a CMV
promoter/enhancer (cf. FIG. 3) was inserted into the AgeI site of
the plasmid 5epocr1000 containing the EPO homology region to obtain
the plasmid p176(cf. FIG. 4a). In order to bring the CMV promoter
as close as possible to the translation start site of the EPO gene,
a 963 bp long segment was deleted between the restriction sites
AscI and AgeI (partial cleavage) to obtain the plasmid p179 (FIG.
4b). 3.2 In order to optimize expression, nucleotides in exon 1
which code for the beginning of the EPO leader sequence
Met-Gly-Val-His were replaced by the synthetic sequence
Met-Ser-Ala-His. This sequence was obtained as a template using
appropriate primers by amplifying a genomic EPO DNA sequence, e.g.,
of the plasmid pEPO148 which contains a 3.5 kB BstEII/EcoRI
fragment (including exons 1-5) of the human EPO gene sequence under
the control of the SV40 promoter (Jacobs et al., Nature 313 (1985),
806 and Lee-Huang et al., Gene 128 (1993), 227). The plasmid p187
was obtained in this process (FIG. 4b). 3.3 The plasmid p189 was
prepared from the plasmid p187 by insertion of the Herpes Simplex
virus thymidine kinase gene (HSV-TK) which was derived from Psvtk-1
(PvuII/NarI fragment) (FIG. 4c). The HSV-TK gene is under the
control of the SV40 promoter and the 3' end of intron 1
(EcoRV/ClaI) in the opposite orientation relative to the CMV
promoter and should serve to negatively select for homologous
recombination. 3.4 For the construction of plasmid p190, a
SfiI/BglII fragment of pHEAVY, a plasmid which contains the cDNA of
an arginine mutant of DHFR described by Simonsen et al. (Proc.
Natl. Acad. Sci. USA 80 (1983), 2495) was subcloned into the
plasmid pGenak-1 cleaved with SfiI and BglII. This plasmid contains
the NEO gene under the control of the RSV promoter and the late
SV40 polyadenylation site as a terminator, the murine DHFR gene
under the control of the early SV40 promoter and the early SV40
polyadenylation site as a terminator (Kaufmann et al., Mol. Cell.
Biol. 2 (1982), 1304; Okayama et al., Mol. Cell. Biol. 3 (1983),
280 and Schimke, J. Biol. Chem. 263 (1988), 5989) and the CMV
promoter (Boshart et al., Cell 41 (1995), 521). Afterwards an HpaI
fragment which contained the cDNA coding for the DHFR arginine
mutant was ligated into the plasmid p189 cleaved with HpaI to
obtain the plasmid p190 (FIG. 4d). 3.5 In order to obtain a
transfection vector without the HSV-TK gene, an AscI/NheI fragment
of the plasmid p190 which contained the gene activation sequence
was ligated into the AscI/NheI fragment of the plasmid p187
containing the exon 1. The resulting plasmid was named p192 (FIG.
4e).
EXAMPLE 4
Transfection of Cells
Various cell lines were selected for the production of EPO and
transfected with targeting vectors.
4.1 Namalwa Cells
Namalwa cells were cultured in T150 tissue culture flasks and
transfected by electroporation (1.times.10.sup.7 cells/800 .mu.l
electroporation buffer 20 mM Hepes, 138 mM NaCl, 5 mM KCl, 0.7 mM
Na.sub.2 HPO.sub.4, 6 mM D-glucose monohydrate pH 7.0, 10 .mu.g
linearized DNA, 960 .mu.F, 260 V BioRad gene pulser). After the
electroporation the cells were cultured in RPMI 1640, 10% (v/v)
fetal calf serum (FCS), 2 mM L-glutamine, 1 mM sodium pyruvate in
forty 96-well plates. After two days the cells were cultured for 10
to 20 days in medium containing 1 mg/ml G-418. The supernatant was
tested in a solid phase ELISA for the production of EPO as
described supra. The EPO producing clones were expanded in 24-well
plates and T-25 tissue culture flasks. Aliquots were frozen and the
cells were subcloned by FACS (Ventage, Becton Dickinson). The
subclones were repeatedly tested for EPO production.
4.2 HT 1080 Cells
The conditions were as described for the Namalwa cells except that
the HT1080 cells were cultured in DMEM, 10% (v/v) FCS, 2 mM
L-glutamine, 1 mM sodium pyruvate. For transfection by
electroporation, cells were detached from the walls of the culture
vessels by trypsinization. After electroporation 1.times.10.sup.7
cells were cultured in DMEM, 10% (v/v) FCS, 2 mM L-glutamine, 1 mM
sodium pyruvate in 5 96-well plates.
4.3 HeLa S3 Cells
Conditions were as described for the Namalwa cells except that the
HeLa S3 cells were cultured in RPMI 1640, 10% (v/v) FCS, 2 mM
L-glutamine, 1% (v/v) NEM non-essential amino acids, 1 mM sodium
pyruvate. For the transfection by electroporation the cells were
detached from the walls of the culture vessels by trypsinization.
The conditions for the electroporation were 960 .mu.F/250 V. After
the electroporation the cells were cultured in RPMI 1640, 10% (v/v)
FCS, 2 mM L-glutamine, 1% (v/v) NEM, 1 mM sodium pyruvate in T75
tissue culture flasks. 24 hours after electroporation the cells
were trypsinized and cultured for 10 to 15 days in a medium
containing 600 .mu.g/ml G-418 in 10 96-well plates.
EXAMPLE 5
Selection of EPO Producing Clones
The culture supernatant of transfected cells was tested in an EPO
ELISA, as described supra. All steps were carried out at room
temperature. 96-well plates pre-coated with streptavidin were
coated with biotinylated anti-EPO antibodies. For coating, the
plates were first washed with 50 mM sodium phosphate pH 7.2, 0.05%
(v/v) Tween 20. Then 0.01 ml coating buffer (4 .mu.g/ml
biotinylated antibody, 10 mM sodium phosphate pH 7.2, 3 g/l bovine
serum albumin, 20 g/l sucrose, 9 g/l NaCl) was added to each well
and incubated for 3 hours at room temperature. Then the plates were
washed with 50 mM sodium phosphate pH 7.2, dried and sealed.
Before the test and after washing three times with 0.3 ml
phosphate-buffered saline (PBS), 0.05% Tween 20 (Sigma), the plates
were incubated overnight with 0.2 ml PBS, 1% (w/v) protein per well
in order to block unspecific binding.
After removing the blocking solution 0.1 ml culture supernatant was
added and the plates were incubated overnight. The individual wells
were each washed three times, with 0.3 ml PBS, 0.05% Tween 20. Then
100 .mu.l peroxidase (POD) conjugated monoclonal anti-EPO antibody
was added for 2 hours. The wells were each subsequently washed,
three times, with 0.3 ml PBS, 0.05% Tween 20. Then the peroxidase
reaction was carried out using ABTS as the substrate in a Perkin
Elmer photometer at 405 nm. A standard calibration curve using
recombinant EPO from CHO cells was used to calculate the EPO
concentrations.
EXAMPLE 6
EPO Gene Amplification
In order to increase the EPO expression, the EPO producing clones
were cultured in the presence of increasing concentrations (100
pM-1000 nM) of methotrexate (MTX). At each MTX concentration the
clones were tested by an ELISA (see example 1.4) for the production
of EPO. Strong producers were subcloned by limiting dilution.
EXAMPLE 7
Signal Sequence Mutations
In order to optimize the leader sequence of the EPO molecule, the
first four amino acids coded by exon 1 were substituted. Primers
with various sequences (SEQ ID NOS: 4-17; the 3' primer contained a
CellI site to select modified sequences) were used to obtain an
AscI/XbaI fragment as a template by PCR using the plasmid pEPO227
which contains a 4 kB HindIII/EcoRI fragment (including exons 1-5)
of the human EPO gene sequence under the control of the SV40
promoter (Jacobs et al., Nature 313 (1985), 806; Lee-Huang et al.,
Gene 128 (1993), 227). The resulting fragments were subsequently
cloned into the plasmid pEPO148 (example 3.2) to obtain the
plasmids pEPO 182, 183, 184 and 185 (FIG. 5). The EPO gene
expression was driven by an SV40 promoter. COS-7 cells were
transiently transfected with the constructs (DEAE-dextan method)
and the cells were tested for EPO production 48 hours after the
transfection.
The mutated leader sequence Met-Ser-Ala-His obtained in this manner
with the best EPO expression was used to construct gene targeting
vectors (cf. example 3.2).
EXAMPLE 8
Characterization of Cell Lines Producing EPO
Three different cell lines (Namalwa, HeLa S3 and HT 1080) were
selected for EPO gene activation. EPO producing clones were
obtained by transfection with the plasmids p179, p187, p189, p190
or p192, described supra.
About 160,000 NEO resistant clones were tested for EPO production,
of which 12-15 secreted EPO reproducibly into the cell supernatant
in significant yield.
Of these clones it was surprisingly possible to identify a total of
7 EPO clones which produced EPO in adequate amounts for a
large-scale production without gene amplification by MTX. This is a
surprisingly high yield. The EPO production of these clones was in
the range of from more than about 200 ng/ml up to more than about
1000 ng/ml/10.sup.6 cells/24 hours.
After gene amplification with 500 nM MTX it was possible to
increase the EPO production of the identified EPO clones to more
than about 3000 ng/ml/10.sup.6 cells/24 hours. Further increase of
the MTX concentration to 1000 nM led to production of more than
about 7000 ng/ml/10.sup.6 cells/24 hours.
The clones obtained produced EPO even under serum-free culture
conditions.
EXAMPLE 9
Characterization of the Genome of the EPO Producing Clones 9.1
Methods
Human genomic DNA was isolated from ca. 10.sup.8 cells and
quantified, following Sambrook et al., supra. After cleavage of the
genomic DNA with restriction enzymes, e.g., AgeI and AscI or BamHI,
HindIII and SalI, the DNA fragments were separated according to
their size by agarose gel electrophoresis and subsequently
transferred and immobilized on a nylon membrane.
The immobilized DNA was hybridized with digoxigenin-labelled
EPO-specific or gene activation sequence-specific DNA probes and
washed under stringent conditions. The specific hybridization
signals were detected with the aid of a chemiluminescent method
using radiation sensitive films.
9.2 Results
The treatment of cells with 500 nM MTX led to an amplification of
the hybridization signal in the EPO locus by a factor of 5 to 10.
When it was increased further to 1000 nM MTX, amplification of
>10 was obtained (FIG. 6a).
In the case of hybridization with an EPO-specific probe, the copies
of the chromosome 7 which were not affected by homologous
recombination were also detected. As can be seen in FIG. 6b, these
DNA fragments which also hybridize have a different size that is
clearly distinguishable and their signal strength was not changed
by the use of MTX.
EXAMPLE 10
Purification of EPO from Culture Supernatants of Human Cell Lines
(HeLa S3; Namalwa and HT1080)
Two methods were used to purify EPO from cell culture supernatants
of human cell lines. These differed in the number and principle of
the chromatography steps and were used depending on the composition
of the medium and the EPO concentrations. In these experiments, EPO
was purified from cell free supernatant of cultured HeLaS3 cells,
where the cell culture medium included 2% (v/v) fetal calf serum.
Method 1: 1st step: blue Sepharose column 2nd step: butyl Sepharose
column 3rd step: hydroxyapatite column 4th step: concentration
Method 2: 1st step: blue Sepharose column 2nd step: hydroxyapatite
column 3rd step: concentration (alternative 3rd step: RP-HPLC)
1. Blue Sepharose Column
A 5 ml blue Sepharose containing column was equilibrated with at
least 5 column volumes (CV) buffer A (20 mM Tris-HCl, pH 7.0; 5 mM
CaCl.sub.2 ; 100 mM NaCl). Subsequently, 70 ml HeLa S3 cell
supernatant (containing ca. 245 .mu.g EPO and 70-100 mg total
protein) was absorbed overnight at a flow rate of 0.5 ml/min in a
circulation process.
The column was washed with at least 5 CVs buffer B (20 mM Tris-HCl,
pH 7.0; 5 mM CaCl.sub.2 ; 250 mM NaCl) and at least 5 CVs buffer C
(20 mM Tris-HCl, pH 7.0; 0.2 mM CaCl.sub.2, 250 mM NaCl) at 0.5
ml/min. The success of the washing was monitored by measuring the
protein content at OD280.
EPO was eluted with buffer D (100 mM Tris-HCl, pH 7.0; 0.2 mM
CaCl.sub.2 ; 2 M NaCl) at a flow rate of 0.5 ml/min. The elution
solution was collected in 1-2 ml fractions.
The EPO content of the fractions, the wash solutions and the flow
through were determined by reverse phase (RP)-HPLC by applying an
aliquot to a column. Alternatively an immunological dot-blot was
carried out for the qualitative identification of fractions
containing EPO.
Fractions containing EPO (8-12 ml) were pooled and applied to a
butyl-Sepharose column.
The yield after the blue Sepharose column was ca. 175 .mu.g EPO
(corresponds to ca. 70%). In general the yield after blue Sepharose
was between 50-75%.
2. Butyl Sepharose Column (Hydrophobic Interaction
Chromatography)
A 2-3 ml butyl Sepharose column was prepared, equilibrated with at
least 5 CV buffer D (100 mM Tris-HCl, pH 7.0; 0.2 mM CaCl.sub.2 ; 2
M NaCl), and subsequently the blue Sepharose pool containing EPO
from 1, supra (ca. 150 .mu.g EPO) was absorbed at a flow rate of
0.5 ml/min.
The column was washed with at least 5 CV buffer E (20 mM Tris-HCl,
pH 7.0; 2 M NaCl and 10% isopropanol) at 0.5 ml/min. The success of
the washing was monitored by measuring the protein content at
OD280.
EPO was eluted with buffer F (20 mM Tris-HCl, pH 7.0; 2 M NaCl and
20% isopropanol) at a flow rate of 0.5 ml/min. The elution solution
was collected in 1-2 ml fractions.
The EPO content of the fractions, the wash solutions and the flow
through were determined by RP-HPLC by applying an aliquot to a
POROS R2/H column. Alternatively, an immunological dot-blot was
carried out for the qualitative identification of fractions
containing EPO.
Fractions containing EPO (10-15 ml) were pooled and applied to a
hydroxyapatite column.
The yield after the butyl Sepharose column was ca. 130 .mu.g EPO
(corresponds to ca. 85%). In general the yield of the butyl
Sepharose was between 60-85% of the applied blue Sepharose
pool.
3. Hydroxyapatite Column
A 5 ml hydroxyapatite column was equilibrated with at least 5 CV
buffer F (20 mM Tris-HCl, pH 7.0; 2 M NaCl; 20% isopropanol) and
subsequently the butyl Sepharose pool containing EPO from 2, supra
(ca. 125 .mu.g EPO) was absorbed at a flow rate of 0.5 ml/min.
The column was washed with at least 5 CV buffer G (20 mM Tris-HCl,
pH 7.0; 2 M NaCl) at 0.5 ml/min. The success of the washing was
monitored by measuring the protein content at OD280.
EPO was eluted with buffer H (10 mM Na-phosphate, pH 7.0; 80 mM
NaCl) at a flow rate of 0.5 ml/min. The elution solution was
collected in 1-2 ml fractions.
The EPO content of the fractions, the wash solutions and the eluant
were determined by RP-HPLC by applying an aliquot to a POROS R2/H
column.
Fractions containing EPO (3-6 ml) were pooled. The yield of the
hydroxyapatite column was ca. 80 .mu.g EPO (corresponds to ca.
60%). In general the yield of the hydroxyapatite column was between
50-65% of the applied butyl Sepharose pool.
4. Concentration
The pooled EPO fractions from the hydroxyapatite step were
concentrated in centrifugation units with an exclusion size of 10
kD to a concentration of 0.1-0.5 mg/ml, admixed with 0.01% Tween 20
and stored in aliquots at -20.degree. C.
Yield Table: EPO (.mu.g) Yield (%) Initial 245 100 blue Sepharose
175 70 butyl Sepharose column 130 53 hydroxyapatite column 80 33
concentration 60 25
The purity of the isolated EPO was about >90%, usually even
>95%.
Method 2 was also used to increase the EPO yield in which the butyl
Sepharose step was omitted. This method can be applied above all to
cell culture supernatants without or with the addition of 1% (v/v)
FCS and yields isolated EPO of approximately the same purity
(90-95%).
The presence of 5 mM CaCl.sub.2 in the equilibration buffer (buffer
F) for the hydroxyapatite column led to improved binding in this
method and thus also to reproducible elution behavior of EPO in the
hydroxyapatite step. Hence, method 2 was carried out with the
following buffers using basically the same process as method 1:
1. Blue Sepharose Column: equilibration buffer (buffer A): 20 mM
Tris-HCl, pH 7.0; 5 mM CaCl.sub.2 ; 100 mM NaCl wash buffer 1
(buffer B): 20 mM Tris-HCl, pH 7.0; 5 mM CaCl.sub.2 ; 250 mM NaCl
wash buffer 2 (buffer C): 20 mM Tris-HCl, pH 7.0; 5 mM CaCl.sub.2,
250 mM NaCl Elution buffer (buffer D): 100 mM Tris-HCl, pH 7.0; 5
mM CaCl.sub.2 ; 2 M NaCl
2. Hydroxyapatite column equilibration buffer (buffer F): 50 mM
Tris-HCl, pH 7.0; 5 mM CaCl.sub.2 ; 1 M NaCl wash buffer (buffer
G): 10 mM Tris-HCl, pH 7.0; 5 mM CaCl.sub.2 ; 80 mM NaCl elution
buffer (buffer H): 10 mM Na phosphate, pH 7.0; 0.5 mM CaCl.sub.2 ;
80 mM NaCl
Yield scheme: EPO (.mu.g) Yield (%) initial 600 100 blue Sepharose
450 75 hydroxyapatite column 335 55 concentration 310 52
The addition of 5 mM CaCl.sub.2 in buffers C, D, E, F, and G in
method 1 also led to improved binding and more defined elution from
the hydroxyapatite column.
EXAMPLE 11
Determination of the Specific Activity in vivo of EPO from Human
Cell Lines (Bioassay on the Normocytaemic Mouse)
The dose-dependent activity of EPO on the proliferation and
differentiation of erythrocyte precursor cells was determined in
vivo in mice via the increase of reticulocytes in the blood after
administration of EPO.
For this, groups of eight mice received various doses of the EPO
sample to be analyzed, and of an EPO standard (matched with the EPO
WHO standard). The mice were subsequently kept under constant
defined conditions. Four days after administration of EPO, blood
was collected from the mice and the reticulocytes were stained with
acridine orange. The reticulocyte number per 30,000 erythrocytes
was determined by microfluorimetry in a flow cytometer by analyzing
the red fluorescence histogram.
The biological activity of the cells was calculated from the values
for the reticulocyte numbers of the sample and of the standard at
the various doses according to the method described by Linder of
paired quantity determination with parallel lines (A. Linder,
"Planen und Auswerten von Versuchen," 3rd edition, 1969,
Birkenhauser Verlag Basel).
Result: specific activity EPO from the cell line U/mg HeLa S3
(sample 1) 100,000 HeLa S3 (sample 2) 110,000
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* * * * *